At 1 month post-injection, haSyn PFF-treated WT and KO-aSyn mice (n = 4/genotype) were perfused transcardially with 0.1M PBS (pH 7.2) at 37°C and then with 4% paraformaldehyde/1% glutaraldehyde. The brain was removed and the ipsilateral striatum was processed for EM analysis as described in Supp. Methods (Online Resource 16) . In all EM procedures, the grids were examined in a Philips 420 transmission electron microscope at an acceleration voltage of 60 kV and photographed with a Megaview G2 CCD camera (Olympus SIS, Münster, Germany) and iTEM image capture software. In order to assess myelin integrity in the PFF-injected mice striatum, we quantified the g-ratio (ratio of inner axonal diameter to total outer diameter) and the % of axons with decompacted myelin [43 (link)]. For these quantifications, at least 100 randomly selected myelin sheaths that were cross-sectioned completely without artifacts and could be classified without doubt were counted. Semi-automated analysis of randomly selected myelin sheaths was carried out using a plug-in for ImageJ software as previously described [7 (link)].
Protocol detail
Myelin Integrity Assessment in Synucleinopathy
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Acceleration
Axons
Brain
Genotype
Glutaraldehyde
Mice
Myelin sheaths
Paraformaldehyde
Striatum
Transmission electron microscope
Corresponding Organization :
Other organizations : Academy of Athens, Biomedical Research Foundation of the Academy of Athens, German Center for Neurodegenerative Diseases, Max Planck Institute for Biophysical Chemistry, University of Florida, Aarhus University, Eginition Hospital, National and Kapodistrian University of Athens
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Variable analysis
independent variables
- Genotype (WT and KO-aSyn)
- G-ratio (ratio of inner axonal diameter to total outer diameter)
- Percentage of axons with decompacted myelin
- Age (1 month post-injection)
- Perfusion conditions (0.1M PBS at 37°C, then 4% paraformaldehyde/1% glutaraldehyde)
- Brain region (ipsilateral striatum)
- EM analysis procedures (Philips 420 transmission electron microscope, Megaview G2 CCD camera, iTEM image capture software)
- Sample size (n = 4/genotype)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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